The present application is a U.S. National Stage Application of PCT/EP 98/07395, filed Nov. 18, 1998. This application also claims the benefit under 35 U.S.C. xc2xa7119 of foreign application nos. EP 97 12 0190.0, filed Nov. 18, 1997 and EP 98 10 3596.7 (Mar. 2, 1998).
The present invention relates to nucleic acid molecules encoding expression products involved in the Responder function, which contributes to the phenomenon of transmission ratio distortion. The present invention also relates to regulatory regions of the genes corresponding to said nucleic acid molecules. The present invention further relates to recombinant DNA molecules and vectors comprising said nucleic acid molecules and/or regulatory regions as well as to host cells transformed therewith. Additionally, the present invention relates to transgenic animals, comprising said nucleic acid molecules, recombinant DNA molecules or vectors stably integrated into their genome. The various embodiments of the invention have a significant impact on breeding strategies by allowing for the specific selection of genetic traits and in particular of sex. Further, the present invention finds applications in the development of contraceptive.
The mouse T/t-complex, a region of approximately 12 cM genetic distance on the proximal part of chromosome 17, contains several loci acting in concert to produce a phenomenon called transmission ratio distortion (TRD). The latter designation indicates the fact that the so-called t-haplotype form of this chromosomal region has a selective advantage over the wild type form in that it is transmitted to the offspring at non-mendelian ratios of up to 99%. This transmission at non-mendelian ratio is achieved by the concerted action of mainly four loci, the so-called Distorters Tcd-1 (D1), Tcd-2 (D2) and Tcd-3 (D3), and the Responder Tcr (Rt)(Lyon 1984). Two more Distorters have been postulated by other authors (Silver and Remis 1987).
According to Lyon""s model (Lyon 1986) which formally explains the genetic interactions of these loci, the Distorters D1, D2 and D3 act strongly and harmfully on the wild type allele of the Responder and weakly on the t form of the Responder (Rt), leading to distortion in favor of Rt. Rt might protect sperm carrying it from this harmful action of the Distorters. The Distorters act in trans while the Responder acts in cis. This means that the chromosome which contains Rt is transmitted at non-mendelian ratio to the offspring. If D2 or all the Distorters are present, the chromosome containing Rt is transmitted at a frequency of more than 50% up to 99% to the offspring. If no Distorter or only D1 or D3 are present, however, the chromosome containing Rt is transmitted at less than 50% to the offspring (xe2x80x9clowxe2x80x9d phenotype). The Distorters are only transmitted at ratios over 50% if they are located on the same chromosome as is Rt. The cis-action of Rt suggests that Rt may be expressed at a stage of spermiogenesis when spermatids are no longer connected in a syncytium (Willison and Ashworth 1987). This would ensure that the product of Rt would be restricted to the spermatozoon containing the t-haplotype form of the R locus. It is expected that expression in elongating spermatids or mature spermatozoa is compatible with this requirement. The trans-acting and cis-acting properties of the Distorters and the Responder, respectively, have been demonstrated by the transmission ratio properties of so-called partial t-haplotypes which carry only a subset of the above named loci (FIG. 1).
Genetic mapping of molecular markers on partial t-haplotypes allowed a more or less precise localization of D1, D2, D3 and Rt to subregions of the T/t-complex and relative to these molecular markers (Lyon 1984; Fox et al. 1985; Herrmann et al. 1986; Silver and Remis 1987; Bullard et al. 1992). Only one locus, Rt could be mapped fairly precisely to a region of appr. 200 kb, the so-called T66B region (Fox et al. 1985; Schimenti et al. 1987; Nadeau et al. 1989; Rosen et al. 1990; Bullard et al. 1992). The T66B region represents a chromosomal piece of the t-haplotype identified by a t-specific restriction fragment length polymorphism detected with the probe Tu66 (Fox et al. 1985). The T66B region is not present in the partial t-haplotypes th44 and th51, but is present in the partial t-haplotypes tlow, th2, th49, t6, and in the complete t-haplotypes, e.g. tw5 or tw12 (FIG. 1). Another partial t-haplotype, tw71Jr1 (abbr. tJr1) contains T66A and a part of T66B. The chromosomes th44, th51and tJr1 do not contain the Rt function, whereas the other partial and complete t-haplotypes named above do. The t-haplotypes containing Rt function must have the t-form of R, whereas the haplotypes th44, th51 and tJr1 are expected to have the wild type form. The genomic region T66B has been cloned molecularly and analyzed. A partial restriction map covering appr. 145 kb of it has been published (Schimenti et al. 1987; Rosen et al. 1990; Bullard et al. 1992).
An extensive and careful search of this region for genes expressed during spermatogenesis has yielded a single gene, T66B-a or Tcp-10bt (Schimenti et al. 1988). Further mapping studies localized xe2x80x9csequences responsible for differential responder activityxe2x80x9d to an interval of 40 kb at the telomeric end of the T66B region which includes Tcp-10bt (Bullard et al. 1992). No other transcription unit could be identified by these authors in the T66B region within the last 10 years. Tcp-10bt has been claimed to represent the candidate for Rt, but a careful analysis showed that it does not encode Responder properties (Schimenti et al. 1988; Cebra-Thomas et al. 1991; Bullard and Schimenti 1990; Ewulonu et al. 1996).
The combined teachings of the prior art thus did not provide any clue how the genetic elements responsible for the Responder phenomenon might be identified. More importantly, the analyses referred to above questioned the prior art discussions that the Responder is a transcription unit. Accordingly, they taught away from the possibility that a transcription unit encoding the Responder might be located in the T66B region. The technical problem underlying the present invention was, accordingly, to overcome these long standing prior art difficulties and provide a genetic entity encoding the Responder function.
The solution to said technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the present invention relates to a nucleic acid molecule comprising a transcription unit encoding in its 5xe2x80x2 portion a kinase having a homology to the MARK2 kinase (Drewes et al., 1997) as well as to other kinases whereas the 3xe2x80x2 portion of the nucleotide sequence has a high homology to the rsk3 kinase (Zhao et al., 1995) as well as to expression products thereof. The term xe2x80x9chomologyxe2x80x9d as used in accordance with the present invention relates to more than 25% and preferably about 38% identity on the amino acid level. Thus, in accordance with the present invention, 38% identity was found in a region of 291 amino acids between MARK2 and the protein encoded by the nucleic acid molecule shown in FIGS. 4a and b or 9a and b. Preferably, the kinase gene encoded by the 5xe2x80x2portion lacks its 3xe2x80x2 end which is preferably an untranslated region whereas the kinase gene encoded by the 3xe2x80x2 portion lacks the 5xe2x80x2 end and is preferably not translated.
Preferably or alternatively, the present invention relates to a nucleic acid molecule encoding an expression product involved in the Responder phenotype, which contributes to the phenomenon of transmission ratio distortion, selected from the group consisting of
(a) a nucleic acid molecule comprising the nucleic acid molecule as shown in FIGS. 4a and b or 9a and b, 7a and b, 7c, d, and e, 7f, g, and h, 7i, j, and k, 7l or a fragment thereof;
(b) a nucleic acid molecule being an allelic variant or a homologue of the nucleic acid sequence of (a);
(c) a nucleic acid molecule hybridizing to a nucleic acid molecule complementary to a nucleic acid molecule of (a) or (b); and
(d) a nucleic acid molecule which is related to the nucleic acid molecule of (a), (b) or (c) by the degeneration of the genetic code.
The term xe2x80x9cResponderxe2x80x9d or xe2x80x9cRxe2x80x9d as used in accordance with the present invention relates to mutant as well as wild type forms of the Responder locus.
The term xe2x80x9cinvolved in the Responder phenotypexe2x80x9d, in accordance with the present invention relates to the fact that transcripts of all genes displayed on FIGS. 4a and b or 9a and b, 7a and b, 7c, d, and e, 7i, j, and k and the antisense transcript of 7f, g, and h are detected in testis carrying complete t-haplotypes, whereas mapping of the genes displayed on FIGS. 4a and b or 9a and b and 7a and b to the t-Responder region suggests that gene 4a and b or 9a and b and/or 7a and b is (are) the one(s) encoding t-Responder activity. In accordance with the further biological data described in this specification, in particular the data relating to the transgenic animals, it is proposed that pursuant to this invention, the gene displayed in FIGS. 4a and b or 9a and b encodes t-Responder activity. The overall data suggest that several genes of the Responder (T66Bk) gene family may act in parallel within t-haplotype carrying spermatids and/or spermatozoa and are thus presumed to be involved in the Responder phenotype, whereby it is envisaged that t-Responder products may antagonize the negative effect of t-Distorter genes and antisense transcripts derived from Responder genes may reduce the activity of Responder genes encoding products with t-Responder as well as wild type or nearly wild-type Responder activity. The latter products may permit the negative action of t-Distorter genes. It is, furthermore, envisaged in accordance with the present invention that alternative translation products from one mRNA-transcript may also be involved in the Responder phenotype (see, e.g., FIGS. 13a and b).
Specifically the cDNA sequence of T66Bk shown in FIGS. 4a and b or 9a and b contains the MARK kinase and the rsk3 kinase homology regions. The cDNA sequence of T66Bk-2 shown in FIGS. 7a and b contains only the MARK kinase homology region. The cDNA sequence of T66k-8 shown in FIGS. 7c, d, and e contains the complete sequence of T66Bk-2 except for a single base deleted between nucleotide position 1508 and 1509 resulting in a frame shift. The cDNA sequence of T66k-7as shown in FIGS. 7f, g, and h corresponds to an antisense transcript of a T66Bk family member. The cDNA sequence of T66k-20 shown in FIGS. 7i, j, and k shows a strong similarity to the above members of the T66Bk gene family.
The term xe2x80x9cfragmentxe2x80x9d as used in connection with the nucleic acid molecule of the invention relates to a fragment that retains the Responder function. Preferably, said fragment comprises the portion of the nucleic acid molecule that has a homology to the MARK kinase referred to above or a part thereof.
As has been indicated above, in one embodiment of the nucleic acid molecule of the invention said expression product is an antisense RNA.
The term xe2x80x9can allelic variant or a homologuexe2x80x9d comprises forms of the wild type or t-allele of the Responder xe2x80x9cgenexe2x80x9d which have been manipulated in vitro in order to achieve an optimal transmission ratio distortion effect and/or to adapt it to the specific requirements of the breeding scheme employed, thus improving the selectability of genetic traits. A number of standard manipulations known in the field are taken into consideration, such as those resulting in the exchange of phosphorylation sites (Ser, Thr, Tyr) on the Responder (poly)peptide for acidic or neutral (Ala) amino acid residues, mutagenesis of the active center, overexpression or knock out mutagenesis of said gene, construction of hypomorphic (poly)peptides by mutagenesis of ATP and/or GTP binding site(s), deletion of phosphorylation sites on said (poly)peptide, deletion of binding sites for other (poly)peptides involved in the Responder/Distorter signaling cascade, synthesis of antisense RNA, N-terminal or C-terminal truncations, introduction of frame shifts which alter part of the amino acid sequence of the protein, etc., resulting either in null, hypomorphic, constitutively active, antimorphic or dominant negative alleles. It is also envisaged that a distortion of the transmission ratio can be achieved with several, if not all, manipulated forms of the Responder gene suggested above. Thus, a manipulated Responder allele affecting the transmission ratio most effectively will have to be identified empirically by employing activity assays in cell culture systems and by employing transgenic animal systems.
It is also envisaged that one or several members of the T66Bk kinase gene family might function as Distorter(s), provided it is (they are) expressed during the diploid or early haploid phase of spermatogenesis allowing distribution of the gene products to all spermatozoa, or can be altered in vitro such as to function as Distorters. The latter may be achieved by in vitro manipulations such as those resulting in the exchange of phosphorylation sites (Ser, Thr, Tyr) on said Responder (poly)peptide for acidic or neutral (Ala) amino acid residues, N- or C-terminal truncation, frame shift, deletion of phosphorylation sites, deletion of binding sites for other (poly)peptides, mutagenesis of the active center, or overexpression of said gene or of antisense transcripts, resulting in constitutively active, hypomorphic, antimorphic or dominant negative gene products and expression of said gene products during the diploid or early haploid phase of spermatogenesis allowing distribution of the gene products to all spermatozoa, e.g. under the control of the Pgk2 promoter. These manipulations are envisaged to have a negative effect on sperm motility and/or fertilization capability. This negative effect may then be balanced by Responder constructs having the opposite effect. The latter could be restricted to those spermatozoa carrying the construct by expressing it under the control of the Responder gene promoter. It is envisaged that both types of spermatozoa would be negatively affected by the Distorter construct expressed in the diploid phase of spermatogenesis, whereas the sperm carrying, in addition, the Responder construct expressed in spermiogenesis would be partially or completely protected by the (poly)peptide expressed in it, and would thus gain an advantage in sperm motility and/or fertilization capability over the other sperm. This would lead to a transmission ratio distortion in favor of the xe2x80x9cprotectedxe2x80x9d spermatozoa. Preferably the Distorter construct expressed in both types of spermatozoa would encode a hypermorphic or constitutively active (poly)peptide, whereas the Responder construct expressed only in those spermatozoa carrying it should encode a hypomorphic, antimorphic or dominant negative (poly)peptide. Both constructs could be integrated on the same or on different chromosomes. Preferably both constructs would be integrated together on the X- or the Y-chromosome, resulting in the preferential or exclusive transmission of the X- or Y-chromosome and thus the preferential or exclusive fathering of female or male offspring, respectively.
The term xe2x80x9chybridizingxe2x80x9d as used in connection with the present invention relates to stringent or nonstringent hybridization conditions. Preferably, it relates to stringent conditions. Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, xe2x80x9cMolecular Cloning, A Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory (1989) N.Y., Ausubel, xe2x80x9cCurrent Protocols in Molecular Biologyxe2x80x9d, Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Hames et al., (eds) xe2x80x9cNucleic acid hybridisation, a practical approachxe2x80x9d IRL Press Oxford, England, (1985).
Stringent hybridization conditions are, for example, hybridization in 6xc3x97SSC, 5xc3x97Denhardt""s reagent, 0,5% SDS, and 100 xcexcg/ml denatured DNA at 65xc2x0 C. and washing in 0,1xc3x97SSC, 0,1% SDS at 65xc2x0 C.
In accordance with the present invention and in contrast to the teachings of the prior art, it was surprisingly found that nucleic acid sequences responsible for the Responder phenotype are comprised at the centromere-close part of the T66 B region. It conforms with several criteria that would be expected for the Responder function:
a) it is located in the T66B region;
b) it is expressed in testis; and
c) it is expressed during spermatogenesis.
In accordance with the present invention, it is further envisaged that additional expression products may contribute to Responder function as has been indicated above which are not necessarily located in the B-region.
As has been indicated above, one of the transcription units (namely T66Bk) contributing to the Responder (R) phenotype apparently arises from two truncated genes. One of said genes has a high homology to the rsk3 gene, the second one has an homology to the MARK kinase recently identified (Drewes et al., 1997). Another transcription unit envisaged to contribute to the R phenotype, T66Bk-2, also has a homology to the MARK kinase, but lacks homology to the rsk3 gene as indicated above. The identification of the genetic basis underlying the R phenotype allows a number of genetic manipulations, in particular in connection with breeding schemes, to be conveniently carried out in the future. Such schemes will be addressed in more detail herein below.
In accordance with the present invention, it is envisaged that the expression products encoded by the nucleic acid sequences of the invention may contribute to the Responder phenotype in several different ways. Thus, in one embodiment one of the above indicated expression products are themselves sufficient to distort the transmission ratio. In another embodiment all of said expression products or combinations of them have to be provided in order to distort the transmission ratio, with certain combinations being more effective than others. In yet another embodiment of the present invention said expression products may work in an additive or synergistic manner. In a still further embodiment it is envisaged that antisense transcripts derived from one or several genes of the T66Bk gene family may contribute to the t-Responder function resulting in a lower level or abolishment of mRNA of one or several T66Bk genes and thus a lower level or abolishment of the corresponding (poly)peptides translated from said mRNA molecules. An example of such an antisense transcript is shown in FIGS. 7f, g, and h. Furthermore, it is suggested that the specifically identified nucleic acid sequences coding for expression products involved in the R phenotype may not be the only ones responsible for the Responder phenotype. Thus, it is envisaged that further nucleic acids encoding expression products that act in concert with the ones discussed above and that may contribute to the Responder phenotype are contained in the genome. Additionally, it is envisaged in accordance with the present invention that the nucleic acid molecules of the invention exert or enhance the Responder phenotype in conjunction with further sequences comprised, for example, in the T66A, T66B and T66C regions. Preferably, said additional regions encode MARK-related kinases.
Also, the person skilled in the art will, on the basis of the teachings of the present invention, be in a position to genetically manipulate the nucleic acid contributing to the Responder phenotype. He will further be in the position to screen the genome of an organism or cell of interest for additional nucleic acid sequences encoding Responder functions on the basis of the genetic organization of the Responder taught in accordance with the present invention. All these embodiments that are without further ado derivable from the specific teachings provided herein are also comprised by the present invention.
It is further envisaged in accordance with the present invention that the Responder may act as a component of a signaling cascade involved in sperm motility and/or the fertilization of oocytes. The t-Responder may act such as to protect the sperm carrying the t-form of the Responder from the negative actions of the t-Distorters whereas the sperm carrying the wild type form of the Responder is xe2x80x9cpoisonedxe2x80x9d (see Lyon 1986). Therefore, the action of the t-form of the Responder somehow counteracts the t-Distorter function suggesting that the Distorters are part of the same signaling cascade. It is, thus, envisaged that the wild type gene or the products of any member of that signaling cascade, once molecularly known, can be manipulated such as to xe2x80x9cpoisonxe2x80x9d the sperm expressing either dominant active or dominant negative forms, or by overexpressing, reducing or abolishing the gene function of any member of said signaling cascade. Selection of genetic traits may then be easily achieved by manipulating the amino acid sequence, activity or expression level of any member of that signaling cascade and restricting the expression of the manipulated form preferentially or completely to those sperm carrying it, such as is the case for the Responder function. The promoter of the Responder or other promoters activating gene expression during the haploid phase of spermatogenesis would be a suitable means for achieving this restriction.
Accordingly, the present invention also relates to methods of influencing transmission ratio by manipulating the expression level or the protein activity of any other member of said signaling cascade. For the purposes of this invention, said cascade is termed xe2x80x9cResponder/Distorter signal cascadexe2x80x9d. It is further envisaged in accordance with the present invention that other signaling cascades may exist besides the Responder/Distorter signaling cascade that may be involved in the motility and/or fertilization capability of spermatozoa. Thus, it is envisaged in accordance with the present invention that the expression level and/or activity of one or more of the proteins involved in said other signaling cascades may be also manipulated in order to influence the transmission ratio. Influencing transmission ratio implies that said ratio may be enhanced or reduced. Methods for manipulating said expression level or said protein activity are known in the art and comprise methods of manipulating amino acid sequences and/or, e.g., promoter strengths or expressing an inhibitor of any member of said signaling cascade. Alternatively, it is envisaged that the expression level may be modulated on the transcription level, the level of pre-mRNA processing, mRNA transport and/or stability, and/or the translation level. Preferably, the modification and/or replacement of elements does not alter the tissue specificity or the specificity for the developmental stage of the expression unit. It is also envisaged in accordance with the present invention that the genetic background of the host organism, the site of integration, and/or the number of integrated copies of a transgene construct may influence the expression efficiency of said transgene construct. Expression or activity of one or more of said members may (significantly) be altered or enhanced, (significantly) be reduced or abolished. Said members also include the Distorters. These methods of the invention can, either alone or in conjunction with other methods described below, advantageously be used for the generation of transgenic animals. Said transgenic animals provide a suitable assay system to test whether the above mentioned methods for manipulating said expression level or said protein activity were successful. Such a system is described in Example 6. Furthermore, said transgenic animals may be employed in any of the breeding schemes addressed below.
In another preferred embodiment of the invention, said nucleic acid molecule is a DNA molecule.
The deduction of the amino acid sequence from the nucleic acid sequence of the invention allows the conclusion that the polypeptide is the expression product that contributes to the Responder phenotype. However, it is not excluded that the mRNA contributes to or triggers said Responder phenotype. Also, it is envisaged in accordance with the present invention that the expression level, stage of expression during spermatogenesis or the copy number of said gene results in or contributes to the Responder phenotype. Therefore, in a preferred embodiment of the nucleic acid molecule of the invention said expression product is an RNA or a (poly)peptide.
A further preferred embodiment of the invention is a nucleic acid molecule, wherein said Responder function is the mouse-t-complex Responder function.
Although it is easily possible to identify mutated or wild-type Responders in animals other than the mouse on the basis of the genetic structure of the Responder that is provided in accordance with the present invention, the mouse t-complex Responder may find applications, for example in breeding, also when introduced into other animals. Specific applications of the Responder function are addressed herein below.
The invention further relates to a regulatory region of the gene corresponding to the nucleic acid molecule of the invention being capable of controlling expression of said nucleic acid molecule.
The term xe2x80x9ccorrespondingxe2x80x9d as used in accordance with the present invention also means that the gene comprises the nucleic acid molecule of the invention or fragments thereof.
The term xe2x80x9cregulatory regionxe2x80x9d in the present application refers to sequences which influence the specificity and/or level of expression, for example in the sense that they confer cell and/or tissue specificity. Such regions can be located upstream of the transcription initiation site, but can also be located downstream of it, e.g., in transcribed leader sequences or in an intron.
The term xe2x80x9ca regulatory region of the gene corresponding to the nucleic acid moleculexe2x80x9d refers to a region with the above mentioned capabilities that controls expression of the bipartite nucleic acid molecule referred to herein also as a xe2x80x9cgenexe2x80x9d.
Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They usually comprise promoters ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers.
Preferably, said regulatory region is a naturally occurring regulatory region or a genetically engineered derivative thereof.
More preferably, said regulatory region comprises or is a promoter. Said promoter is preferably tissue specific and confers expression, for example, during spermiogenesis.
The term xe2x80x9cpromoterxe2x80x9d refers to the nucleotide sequences necessary for transcription initiation, i.e. RNA polymerase binding, and also includes, for example, the TATA box.
In one embodiment, said promoter is or comprises a minimal promoter. According to the present invention, promoters from other species can be used that are functionally homologous to the regulatory sequences or the promoter of the murine gene, or promoters of genes that display an identical pattern of expression, in the sense of being expressed in sperm cells. As has been outlined above, it is possible for the person skilled in the art to isolate with the help of the known murine nucleic acid corresponding genes from other species, for example, human. This can be done by conventional techniques known in the art, for example, by using the nucleic acid molecule of the invention as a hybridization probe or by designing appropriate PCR primers. It is then possible to isolate the corresponding promoter region by conventional techniques and test it for its expression pattern. For this purpose, it is, for instance, possible to fuse the promoter to a reporter gene, such as the lacZ gene or green fluorescent protein (GFP) and assess the expression of the reporter gene in transgenic mice.
The present invention also relates to the use of promoter regions which are substantially identical to the murine promoter or to a promoter of a homologous gene or to parts thereof and which are able to confer specific expression in sperm cells.
Such promoters differ at one or more positions from the above-mentioned promoters but still have the same specificity, namely they comprise the same or similar sequence motifs responsible for the above described expression pattem. Preferably such promoters hybridize to one of the above-mentioned promoters, most preferably under stringent conditions. Particularly preferred are promoters which share at least 85%, more preferably 90-95%, and most preferably 96-99% sequence identity with one of the above-mentioned promoters and have the same specificity. Such promoters also comprise those which are altered, for example by deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination in comparison to the above-described nucleotide sequence. Methods for introducing such modifications in the nucleotide sequence of the promoter of the invention are well known to the person skilled in the art. It is also immediately evident to the person skilled in the art that further regulatory sequences may be added to the promoter of the invention. For example, transcriptional enhancers and/or sequences which allow for induced expression of the promoter of the invention may be employed. A suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62).
Most preferably, said regulatory region comprises the fragment from nucleotides 930 to 3576 of the sequence shown in FIG. 11.
Also comprised are fragments or variants of the above sequence wherein the regulatory function of said fragments or variants is essentially retained or even improved. This may be tested according to methods well known in the art in combination with the teaching of this specification.
The invention further relates to a recombinant DNA molecule comprising a nucleic acid molecule of the invention and/or a regulatory region of the invention and/or a regulatory region allowing expression during spermatogenesis/spermiogenesis.
Accordingly, the regulatory region may control expression of the nucleic acid molecule contributing to the Responder function. Alternatively, said recombinant DNA molecule may comprise said regulatory region which controls expression of a heterologous nucleic acid or which is not operatively linked to any nucleic acid and, thus, may be used for cloning purposes. In the first alternative, said regulatory region is operatively linked to a heterologous DNA sequence. For example, said regulatory region may be operatively linked to a naturally occurring or in vitro engineered DNA encoding a member of the Responder/Distorter cascade, for example, a Distorter or a member of another signaling cascade involved in sperm motility and/or fertilization. Also, in this embodiment of the invention, the nucleic acid molecule of the invention may be operatively linked to a different or to no regulatory region. The regulatory region may be the original regulatory region of the gene corresponding to the nucleic acid molecule of the invention or may be derived from a different copy of said gene or from a different gene. Furthermore, the regulatory region may be derived from a copy of the homologous gene (in case more than one copy exists) from a different species or may be derived from a different gene from said different species. The above-mentioned regulatory regions may also be modified in order to obtain optimum expression, which may be enhanced or reduced expression. Thus, it is envisaged in accordance with the present invention that e.g., the regulatory regions controlling expression of the gene comprising the T66k-20-cDNA (see FIGS. 7i, j, and k) or the cDNAs shown in FIG. 10 are used in unmodified or modified form in accordance with the present invention. Due to the teaching of the present invention, namely the cloning and the disclosure of the sequences of the cDNAs, it is routine experimentation for the person skilled in the art to clone and use said regulatory regions.
Advantageously, the recombinant DNA molecule of the invention may further comprise an expression unit encoding and expressing a desired genetic trait. Such a DNA molecule may be used to reduce, or enhance the inheritance of said desired genetic trait, provided that either the recombinant DNA molecule further comprises an expression unit encoding and expressing at least one Distorter or protein with Distorter activity, preferably D2, or the genetic background of the host provides such Distorter activity which may be naturally occurring in said host or which may have been introduced.
A particularly preferred embodiment of the invention relates to a recombinant DNA molecule, wherein said heterologous DNA sequence encodes a peptide, protein, antisense RNA, sense RNA and/or ribozyme. As regards the antisense RNA, it may find applications in methods of antisense therapy or antisense knockout strategies. Antisense therapy may be carried out by administering to an animal or a human patient, a recombinant DNA containing the regulatory sequences of the invention operably linked to a DNA sequence, i.e., an antisense template which is transcribed into an antisense RNA. The antisense RNA may be a short (generally at least 10, preferably at least 14 nucleotides, and optionally up to 100 or more nucleotides) nucleotide sequence formulated to be complementary to a portion of a specific mRNA sequence. Standard methods relating to antisense technology have been described (Melani, Cancer Res. 51 (1991), 2897-2901). Following transcription of the DNA sequence into antisense RNA, the antisense RNA binds to its target mRNA molecules within a cell, thereby inhibiting translation of the mRNA and down-regulating expression of the protein expected to be encoded by the mRNA. For example, an antisense sequence will be complementary to a portion of or all of the mRNA. In addition, ribozymes may advantageously be employed to eliminate wild-type Responder transcripts from cells.
The invention further relates to a recombinant DNA molecule, wherein said peptide, protein, antisense RNA, sense RNA, a toxin and/or ribozyme is capable of causing cell death.
In this embodiment of the invention, sperm which do not carry the R related transgene can be genetically selected.
For example, the promoter of the R gene can be used for the expression of a gene product inducing the destruction or apoptosis of said spermatocytes carrying said construct. Integration of such a construct on the X- or Y-chromosome will result in the transmission of the respectively other sex chromosome. Integration of the construct on the X chromosome will lead to the neutral transmission of the construct in female animals. Integration in the Y chromosome should, preferably, be in an inactive state that can be activated along the rules that will be laid down herein below.
A recombinant DNA molecule which further comprises DNA encoding an effector polypeptide is a further preferred embodiment of the invention.
It is particularly preferred that said effector polypeptide is capable of sequestering an ion selectively binding to a solid support, or binding to a preselected antigenic determinant or is a toxin, an enzyme, a ribozyme, a label or a remotely detectable moiety.
In accordance with the invention, it is most preferred that said effector polypeptide is calmodulin, methallothionein, a fragment thereof, green fluorescent protein (GFP), xcex2-lactamase (Zlokamik et al., 1998), hCD24, myc, FLAG, hemagglutinin or an amino acid sequence rich in at least one of glutamic acid, aspartic acid, lysine, histidine or arginine.
Accordingly and in other words, the above embodiments of the invention relate to the use of the R promoter for the expression of a (poly)peptide being or having a tag. Said tag may be expressed in the cytoplasm of sperm. An example of such a tag is GFP or xcex2-lactamase. Said tag is alternatively located on the surface of sperm and thus, may be recognized by specific antibodies. This enables the separation of sperm carrying a transgene expressed under the control of the R promoter from sperm not carrying said transgene. The person skilled in the art is familiar with a variety of methods for the separation of sperm carrying said tag on its surface. Preferably, said tag is selected by affinity chromatography or by using a cell sorter. After separation, sperm carrying the transgene or sperm without the transgene can be used for fertilization of eggs. This embodiment includes integration of transgene in either autosomes or sex chromosomes. Advantageously, the solid support referred to above is a membrane or the surface of an ELISA plate.
Further, the invention relates to a vector comprising the nucleic acid molecule of the invention, the regulatory region of the invention or the recombinant DNA molecule of the invention.
The vector of the invention may simply be used for propagation of the genetic elements comprised therein. Advantageously, it is an expression vector and/or a targeting vector. Expression vectors such as Pichia pastoris derived vectors or vectors derived from viruses such as CMV, SV-40, baculovirus or retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the recombinant DNA molecule or vector of the invention into targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, loc. cit. and Ausubel, loc. cit. Alternatively, the recombinant DNA molecules and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
It is preferred according to one further embodiment that said vector comprises a heterologous promoter.
Said heterologous promoter not naturally operatively linked with the nucleic acid contributing to the Responder function may be used to determine a certain time point of the onset of Responder expression. This time point may be the same or a different one that is set when the natural Responder transcription unit is employed. For example, said heterologous promoter may also be active in the early or late haploid phase of spermatogenesis.
It is particularly preferred that said heterologous promoter is controlling gene expression in spermatogenesis and/or in spermiogenesis.
Most preferably, said heterologous promoter is the testis promoter of c-kit or of Angiotensin-Converting-Enzyme (ACE), both of which are well known in the art.
The invention further relates to a host cell transformed or transfected with the nucleic acid molecule, the recombinant DNA molecule or the vector of the invention. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell. Prokaryotic host cells will usually only be employed for the propagation of the nucleic acid molecule of the invention and sometimes for the production of the expression product. Suitable mammalian, fish or bird cell lines are well known or can easily be determined by the person skilled in the art and comprise COS cells, Hela cells, primary embryonic cell lines etc.
The term xe2x80x9ctransfected or transformedxe2x80x9d is used herein in its broadest possible sense and also refers to techniques such as electroporation, infection or particle bombardment.
Furthermore, the invention relates to a method of recombinantly producing an expression product as defined herein above comprising the steps of culturing the host cell of the invention under conditions to cause expression of the protein and recovering said protein from the culture.
The method of the invention is most advantageously carried out along conventional protocols which have been described, for example, in Sambrook, loc. cit.
The invention further relates to an expression product encoded by the nucleic acid molecule of the invention or which is obtainable by the production method of the invention.
In accordance with the invention, said expression product may either be an mRNA or a polypeptide. Said expression product is, in accordance with the present invention, involved in the Responder phenotype and contributes to the phenomenon of transmission ratio distortion.
A further embodiment of the invention relates to an antibody specifically recognizing the expression product of the invention.
The antibody of the invention may be a monoclonal antibody or an antibody comprised in a polyclonal serum. Accordingly, the term xe2x80x9cantibodyxe2x80x9d as used herein also relates to a polyclonal antiserum. In addition, said term relates to antibody fragments or fusion proteins comprising antibody binding sites such as Fab, Fv, scFv fragments etc. The antibody of the invention has a number of applicabilities including purification or diagnostic processes.
The invention additionally relates to a nucleic acid molecule specifically hybridizing with the nucleic acid molecule of the invention translatable into said MARK related kinase or to an intron of said nucleic acid molecule or with the regulatory region of the invention or with a complementing strand thereof.
Said nucleic acid molecules comprise at least 15 nucleotides in length and hybridize specifically with a nucleic acid or regulatory sequence as described above or with a complementary strand thereof. Specific hybridization occurs preferably under stringent conditions and implies no or very little cross-hybridization with nucleotide sequences having no or substantially different regulatory properties. Such nucleic acid molecules may be used as probes and/or for the control of gene expression. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary in length. Preferred are nucleic acid probes of 17 to 35 nucleotides in length. Of course, it may also be appropriate to use nucleic acids of up to 100 and more nucleotides in length. The nucleic acid probes of the invention are useful for various applications. On the one hand, they may be used as PCR primers for amplification of regulatory sequences according to the invention. In this embodiment, one of the primers may hybridize to the 3xe2x80x2 portion of the Responder having a high homology to the rsk3 gene. Another application is the use as a hybridization probe to identify regulatory sequences hybridizing to the regulatory sequences of the invention by homology screening of genomic DNA libraries. Nucleic acid molecules according to this preferred embodiment of the invention which are complementary to a regulatory sequence as described above may also be used for repression of expression of a gene comprising such regulatory sequences, for example due to an aniisense or triple helix effect or for the construction of appropriate ribozymes (see, e.g., EP-B1 0 291 533, EP-A1 0 321 201, EP-A2 0 360 257) which specifically cleave the (pre)-mRNA of a gene comprising a regulatory sequence of the invention. Selection of appropriate target sites and corresponding ribozymes can be done as described for example in Steinecke, Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds Academic Press, Inc. (1995), 449-460. Furthermore, the person skilled in the art is well aware that it is also possible to label such a nucleic acid probe with an appropriate marker for specific applications, such as for the detection of the presence of a nucleic acid molecule of the invention in a sample derived from an organism.
The above described nucleic acid molecules may either be DNA or RNA or a hybrid thereof. Furthermore, said nucleic acid molecule may contain, for example, thioester bonds and/or nucleotide analogues, commonly used in oligonucleotide anti-sense approaches. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell. Said nucleic acid molecules may be transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell. Such nucleic acid molecules may further contain ribozyme sequences which specifically cleave the (pre)-mRNA comprising the regulatory sequence of the invention. Furthermore, oligonucleotides can be designed which are complementary to a regulatory sequence of the invention (triple helix; see Lee, Nucl. Acids Res. 6 (1979), 3073; Cooney, Science 241 (1988), 456 and Beal et al., Science 251 (1991), 1360), thereby preventing transcription and the production of the encoded mRNA and/or protein.
Furthermore, the invention relates to a pharmaceutical composition comprising the DNA molecule, the regulatory region, the recombinant DNA, the vector, the host cell, the expression product or the antibody of the invention.
Said pharmaceutical composition comprises at least one of the aforementioned compounds of the invention, either alone or in combination, and optionally a pharmaceutically acceptable carrier or excipient. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by conventional methods. These pharmaceutical compositions can be administered to subject in need thereof at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient""s size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 xcexcg (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 xcexcg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 xcexcg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 106 to 1022 copies of the nucleic acid molecule. The compositions of the invention may be administered locally or systematically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer""s dextrose, dextrose and sodium chloride, lactated Ringer""s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer""s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition.
It is envisaged by the present invention that in particular the various recombinant nucleic acid/DNA molecules and vectors of the invention are administered either alone or in any combination using standard vectors and/or gene delivery systems, and optionally together with an appropriate compound and/or together with a pharmaceutically acceptable carrier or excipient. Subsequent to administration, said molecules may be stably integrated into the genome of the mammal, fish or bird. On the other hand, viral vectors may be used which are specific for certain cells or tissues, preferably for pancreatic cells and persist in said cells. Suitable pharmaceutical carriers and excipients are well known in the art.
The invention further relates to a diagnostic composition comprising the nucleic acid molecule, the regulatory region, the recombinant DNA molecule, the vector, the host cell, the expression product or a primer or an oligonucleotide hybridizing to the nucleic acid molecule or regulatory region of the invention or to a complementary strand thereof and preferably to the regions identified herein above or the antibody of the invention. Comprised by the above definition of the term xe2x80x9cprimerxe2x80x9d are also pairs of primers such as forward and reverse primers that may be used for PCR. One of said primers of said pair of primers may hybridize in the region of the rsk-related nucleic acid sequence.
In one embodiment, said diagnostic composition is manufactured in the form of a kit.
Said compositions may additionally contain further compounds such as plasmids, antibiotics and the like for screening animals or cells for the presence of nucleic acid sequences or regulatory elements corresponding to those identified in the appended examples or described herein above. The components of the diagnostic composition and/or kit of the present invention may be packaged in containers such as vials, optionally in buffers and/or solutions. If appropriate, one or more of said components may be packaged in one and the same container. Additionally or alternatively, one or more of said components may be adsorbed to a solid support such as, e.g., a nitrocellulose filter or nylon membrane, or to the well of a microtiter plate.
The invention further relates to a method for the production of a transgenic non human mammal, fish or bird comprising introducing the nucleic acid molecule, the regulatory region, the recombinant DNA molecule or the vector of the invention into a cell, preferably germ cell, embryonic cell or an egg cell or a cell derived therefrom.
Methods for the generation of such transgenic animals are well known in the art and are described, for example, in xe2x80x9cGuide to techniques in mouse developmentxe2x80x9d (ed. Wassarman and DePamphilis) Methods in Enzymology Vol. 225 (Academic Press, 1993). The method of the invention also comprises embodiments related to the cloning of such animals. These embodiments include the steps of introducing said nucleic acid molecule, recombinant DNA molecule or vector of the invention into the nucleus of a cell, preferably an embryonic cell, replacing the nucleus of an oocyte, a zygote or an early embryo with said nucleus comprising said nucleic acid molecule, recombinant DNA molecule or vector of the invention, transferring either said ooyte, zygote or early embryo into a foster mother or first in vitro or in vivo culturing said oocyte, zygote or early embryo and subsequently transferring the resulting embryo into a foster mother and allowing the embryo to develop to term; see, for example, Wilmut I. et al. (1997) xe2x80x9cViable offspring derived from fetal and adult mammalian cellsxe2x80x9d, Nature 385, 810-813.
In a preferred embodiment of the method of the invention, said chromosome is an X chromosome or the corresponding sex chromosome in birds or fish or an autosome.
In an alternative preferred embodiment of the method of the invention, said chromosome is a Y chromosome, or the corresponding sex chromosome in birds or fish.
It is particularly preferred that the nucleic acid molecule, the regulatory region, the recombinant DNA molecule or the vector of the invention, a heterologous promoter controlling expression in spermiogenesis and/or a DNA sequence encoding an effector (poly)peptide as defined hereinabove alone or in combination is/are integrated in said Y chromosome in a reversible inactive state of expressibility.
In accordance with the method of the invention, it is most preferred that said nucleic acid molecule, regulatory region, recombinant DNA molecule, vector of the invention, a heterologous promoter controlling expression in spermiogenesis and/or a DNA sequence encoding an effector (poly)peptide as defined hereinabove alone or in combination is/are flanked by lox P sites or FRT sites.
In all the above embodiments, at least one Distorter may be present on the same or on different chromosome.
An additional particularly preferred embodiment of the method of the invention further comprises introducing a nucleic acid molecule encoding at least one Distorter into the same or a different chromosome or introducing a chromosomal fragment comprising at least one Distorter into said cell. Advantageously, said Distorters are the mouse t-complex Distorter loci.
It is most preferred that said Distorter is/are D2 and/or D1.
Said method of the invention and its various preferred embodiments provide a wide range of applications in particular in the breeding of animals. Thus, as has been outlined above, the nucleic acid sequence encoding a molecule contributing to the Responder and/or an effector (poly)peptide as defined hereinabove may be under the regulation of the promoter naturally associated with said nucleic acid sequence. Integration of such a construct into a chromosome will, in the absence of a Distorter function result in a disadvantage in a chromosome if it comes to transmission of said chromosome. This disadvantage may be in the range of 49 to 0% transmission ratio. In the case that the Responder effect results in a very low or no transmission of the corresponding chromosome and if, in addition, the above recited construct comprising the nucleic acid molecule of the invention or the effector (poly)peptide is integrated into the Y chromosomes, the Y chromosome and the Responder function would hardly or not be transmitted by male animals. In order to provide for male animals, the Y chromosome should advantageously comprise an inactive construct that can, however, be activated. Said inactive construct should be without influence on the transmission ratio. One embodiment of said construct comprises loxP or FRT sites which flank an intervening sequence located between said promoter or a heterologous promoter controlling expression in spermiogenesis and effector (poly)peptide encoding sequences and/or sequences conferring Responder activity. The intervening sequence would be designed in such a way as to prevent the expression of effector and/or Responder activity. Activation of the effector and/or Responder activity may be effected by excision of the intervening sequence due to activity of the Cre or flp protein comprised in the same cell. Another embodiment of said construct comprises loxP or FRT sites flanking said promoter or a heterologous promoter controlling expression in spermiogenesis whereby the promoter is oriented away from the construct comprising the nucleic acid of the invention or the effector sequences encoding the above mentioned (poly)peptides. The activity of Cre or flp would allow the promoter to be inverted resulting in the transcription of the effector sequences or the sequences contributing to Responder activity during spermiogenesis. Another embodiment of said construct comprises loxP or FRT sites flanking said nucleic acid sequences reversely oriented towards the promoter such that the antisense strand is transcribed during spermiogenesis. Activation may be effected by flipping the effector sequences or the sequences contributing to Responder activity due to the activity of Cre or flp comprised in the same cell. Expression of the Cre or flp protein would advantageously be effected prior to spermiogenesis. The activation of the Responder or effector function is in such cases effected during spermatogenesis under the control of the R promoter or another promoter controlling expression during spermatogenesis/spermiogenesis. Preferably, the Cre gene is integrated on an autosome and may be expressed under the control of one of the following promoters: cytomegalovirus immediate early enhancer-chicken beta-actin hybrid (CAG) promoter, wherein site specific recombination occurs in the zygote; adenovirus Ella promoter, wherein expression is triggered during early embryogenesis; CMV, wherein expression is triggered during embryogenesis; OCT4, wherein expression is also triggered during embryogenesis and in germ line cells; HSV-TK or Pgk, wherein expression is ubiquitous; or Pgk2, wherein the construct is expressed during spermatogenesis. In the above embodiment, the Responder and/or effector encoding construct is transmitted by male animals in an inactive state. Mating with a female carrier of the Cre construct will result in male progeny having their Responder and/or effector activated during spermatogenesis. Progeny of these male animals inherit predominantly or exclusively the X chromosome of the father and are accordingly female progeny. In the case that the X chromosome is exclusively transmitted, the Responder and/or effector function is not inherited by the progeny. However, in cases of a less strong effect of the Responder and/or effector (poly)peptide leading to, for example, 10 to 20% transmission, the inactivation of the construct is not necessary because this low transmission is sufficient for the generation of male carriers. The frequency of inheritance of the R gene of the mouse, without the interaction of t-Distorters, is naturally in the range of about 20%.
In an alternative preferred embodiment of the method of the invention that has been identified above, the Responder and/or effector is integrated on the X chromosome or on an autosome. In this case, no inactive construct is necessary, since the Responder and/or effector encoding construct is transmitted in female animals in a neutral state, because Responder function only acts during spermatogenesis. Mating with wild type male animals leads to the generation of male animals carrying an active R and/or effector encoding gene on the X chromosome or an autosome. The chromosome carrying the R and/or effector encoding gene has a disadvantage in transmission. This means less than 50% to 0% of the progeny inherit said chromosome. In the case that the R and/or effector encoding construct is integrated into the X chromosome, no female progeny or only a low percentage of female progeny will be generated.
Furthermore, the invention relates to a method for the production of a male transgenic non human mammal, fish or bird having integrated in its Y or corresponding sex chromosome the nucleic acid molecule, the regulatory region, the recombinant DNA molecule or the vector of the invention, a heterologous promoter controlling expression in spermiogenesis and/or a DNA sequence encoding an effector (poly)peptide as defined hereinabove alone or in combination in an active state of expressibility, said method comprising in vitro fertilization or injection of spermatozoa into eggs using sperm from said male transgenic non human mammal, fish or bird. In a preferred embodiment of the present invention, said method prior to in vitro fertilization or injection further comprises allowing expression of said effector (poly)peptide and selecting for sperm expressing said effector (poly)peptide and, thus, containing said Y or corresponding sex chromosome. The above method is useful in case the transmission of the construct from male carriers by natural mating or artificial insemination is close to 0%. The production of transgenic male carriers can be achieved by the method of the invention using in vitro fertilization since it has been shown in mice that transmission ratio distortion of t/+ sperm does not occur during in vitro fertilization. The efficiency of the method of the invention can be further enhanced by selection for sperm carrying a Y or corresponding sex chromosome prior to in vitro fertilization as described above. Selection can be effected, e.g., by cell sorting.
Alternatively, male carriers of the R and/or effector function which are used for the generation of predominantly female progeny result from mating of hemizygous male animals carrying an inactive R and/or effector encoding construct with hemizygous female animals carrying a locus encoding a site specific recombinase and preferably the Cre locus. Progeny of such matings may be used for the maintenance of the strain as well as for the generation of the desired female progeny. It is worthwhile noting that from a single male carrier of the R and/or effector encoding construct many female progeny can be obtained.
A further embodiment of the invention that has been referred to above relates to the use of the R gene in combination with Distorter 2 (D2) preferably in combination with Distorter 1 (D1). In this embodiment, the chromosome carrying the R construct is transmitted predominantly or exclusively.
Distorters D1 and D2 (and possibly D3 as well as further postulated Distorters) act in trans to the advantage of the chromosome carrying the R construct. Whereas the applicant does not wish to be bound by any scientific theory, it is presently assumed that D1 and D2 are expressed in the diploid phase of spermatogenesis. Whereas the Distorter genes have not yet been identified it is well known that their gene products lead to the predominant or exclusive transmission of the chromosome carrying the R function. The Distorter function can be provided, for example, by a chromosome carrying a partial t-haplotype containing, e.g., Distorter D1 or D2 or both. It is further presumed that the expression products of the Distorter genes exert a negative influence on sperm not carrying the R function. In contrast, the sperm carrying the R function are protected by the R function. It is also suggested that such sperm may have a selective advantage as regards motility and thus faster reach the egg cell to be fertilized.
It is envisaged in accordance with the present invention that D2, D1 and further Distorters are located on the same or one or more different chromosomes than that or those which carry/carries the R construct. If R is integrated on the Y chromosome, mating will predominantly result in male progeny. Integration on the X chromosome, in contrast, will yield predominantly or exclusively female progeny. Integration in an autosome will result in a high transmission of said chromosome and thus any trait linked to said R construct. The high transmission of the R construct guarantees the maintenance of the R function. A practical advantage of the embodiment, in the case that the R encoding construct is integrated in the X chromosome, is that only few male wild type animals are necessary for the maintenance of the Y chromosome, i.e., of the male sex. Said male wild type animals may be generated by mating transgenic hemizygous female animals, carrying both the Distorter(s) and the R function with wild type males.
The subject-matter of the invention relates also to a transgenic non human mammal, fish or bird having stably integrated in its genome the nucleic acid molecule, the regulatory region, the recombinant DNA molecule or the vector of the invention or which is regenerated from a host cell of the invention or which is obtainable by the method of the invention referred to above.
Said transgenic animal is advantageously mouse, cattle, sheep, pig, goat, rat, rabbit, horse, dog, cat, camel, chicken, duck, salmon or trout.
Said transgenic animals may be used for producing offspring at a non mendelian ratio comprising breeding, in vitro fertilization or artificial insemination.
The invention additionally relates to a pair of transgenic non human mammals, fish or bird, wherein the male is a transgenic animal having integrated in its Y chromosome the nucleic acid molecule, the regulatory region, the recombinant DNA molecule, or the vector of the invention in a reversible inactive state of expressibility and optionally at least one Distorter in its genome, and the female is a transgenic animal having stably integrated into its genomic DNA a nucleic acid molecule encoding a site specific DNA recombinase.
The pair of transgenic animals should of course be preferably of the same species in order to allow a successful mating.
Preferably, in said female of said pair of animals, said DNA recombinase is Cre or flp.
Most advantageously, said DNA recombinase is controlled by regulatory elements that are active prior to spermiogenesis.
Further, the present invention relates to sperm obtainable from a male of the transgenic non-human mammal, fish or bird as defined herein before. Said sperm may be comprised in a composition suitable, for example, for deep freezing.
The invention also relates to a method for the selection of the sperm of the invention comprising allowing expression of the effector (poly)peptide and selecting for the presence or absence of said (poly)peptide.
In accordance with this method of the invention, the effector (poly)peptide is preferably selected for by cell sorting or affinity chromatography. Sperm either carrying or not carrying the effector (poly)peptide and thus the nucleic acid molecule of the invention may then be used for the further desired purpose.
Additionally, the invention relates to a method for the selection against sperm of the invention comprising
(a) allowing expression of the recombinant DNA molecule defined herein above that is capable of causing cell death; and
(b) selecting for viable sperm.
Cell death can advantageously also be caused by the in vivo expression of an effector molecule comprising a tag and the addition of a specific antibody binding to the tag and of complement to sperm in vitro, resulting in the inactivation or lysis of the spermatozoa carrying the construct.
Said methods find applicability in cases where sperm carrying the R promoter function is to be selected against.
A further object of the invention is the use of the sperm for the production of offspring. Such a production may comprise breeding, in vitro fertilization or artificial insemination.
An additional object of the present invention relates to the use of the nucleic acid molecule of the invention, the regulatory region of the invention, the recombinant DNA of the invention, the vector of the invention, the host cell of the invention, the expression product of the invention or the antibody of the invention for the isolation of receptors on the surface of sperm recognizing attractants of the egg cell for the development and/or production of contraceptive.
Further, the present invention relates to the use of the nucleic acid molecule of the invention, the regulatory region of the invention, the recombinant DNA of the invention, the vector of the invention, the host cell of the invention, the expression product of the invention or the antibody of the invention for the identification of chemicals or biological compounds able to trigger the (premature) activation or inhibition (repression) of the signaling cascade in which the Responder function is envisaged to be involved in. Such compounds could be applicable as potent contraceptiva since it is envisaged that the activation or inhibition (repression) of said signaling cascade may affect the motility of sperm, due to rapid exhaustion of their energy reserve, and/or by inhibiting sperm movement and/or affect the ability of sperm to fertilize ovulated eggs.
The identification of said chemical or biological compounds could be achieved by standard screening technology using the activation of the wild type Responder protein expressed in cell culture cells as an assay. It is e.g. envisaged that activation of said protein may trigger microtubule disruption in cell culture cells similar to the effect obtained by overexpression of the MARK kinase. Compounds triggering or inhibiting such an effect could then be tested for their effect on the motility and/or fertilization ability of sperm. Alternatively, a similar screening system for said compounds could also be envisaged for sperm without prior employment of a screening assay in cell culture cells.
Furthermore, the nucleic acid molecule of the invention, the regulatory region of the invention, the recombinant DNA of the invention, the vector of the invention, the host cell of the invention, the expression product of the invention or the antibody of the invention can be used for the isolation of receptor molecules and/or other members of the Responder/Distorter signaling cascade to which said expression product which would be expected to be a (poly)peptide may bind. Said signal transducing molecules may be identified by immunoprecipitation of protein complexes involving the Responder (poly)peptide and cloning of the corresponding genes encoding them, or by Two Hybrid Screening techniques in yeast employing standard technology. In particular, most preferably the Responder gene or (poly)peptide may be used to isolate the membrane receptor of the signaling molecule which is envisaged to activate said Responder/Distorter signaling cascade. Said membrane receptor is envisaged to be most preferable as a target for the development of novel contraceptives.
Additionally, the present invention relates to a method for the detection of the nucleic acid molecule, the regulatory region, the recombinant DNA molecule, the vector, or the expression product of the invention or a different heterologous expression product encoded by said DNA molecule or vector in the transgenic non human mammal, fish or bird of the invention or a part thereof comprising identifying said nucleic acid molecule, regulatory region, recombinant DNA molecule or vector of the invention or a portion thereof in said transgenic animal or said part thereof. The method of the invention allows the identification of animals of the invention on the basis of the genetic constructs they carry in accordance with the invention. Moreover, the method allows the identification of such animals e.g. after slaughtering by analyzing parts thereof. It should be noted that sperm, egg cells and embryos are also to be considered as parts of said animals. Detection may be effected by PCR using primers specified herein above. Nucleic acid hybridization with a detectably labeled probe constitutes a different method of detection. It is further most important to note that any portion or component of the nucleic acid, recombinant DNA molecule or vector may be identified in accordance with the method of the invention as long as it is indicative thereof. Thus, for example, the vector may comprise a nucleic acid sequence without any biological function that is nevertheless indicative of said vector and thus, of the invention. In another embodiment the effector (poly)peptide may be used for detection. Of course, the nucleic acid molecule of the invention or a portion thereof may itself be detected. All embodiments conceivable by the person skilled in the art that comprise the above step underfall the method of the invention as long as they allow the detection of the above mentioned genetic material.
Also, the present invention relates to a method of distorting the transmission ratio of genetic traits comprising manipulating the sequence or expression level of a different member of the Responder/Distorter signal cascade than the t-Responder, and restricting the expression of the manipulated form of said different member preferentially or completely to those sperm carrying it.
Preferred embodiments and various applications of this method as well as methods of manipulating said sequence or expression level have been addressed herein before.
The invention also relates to a transgenic animal having a recombinantly manipulated altered sequence or expression level of a member of the Responder/Distorter signal cascade, and wherein the expression of said member has been restricted preferentially or completely to those sperm carrying it.
Preferably, said member of said signal cascade is not the Responder.
In these embodiments of the invention, the sequence or expression level of a preferably different member of the cascade than the Responder is altered or abolished. Simultaneously, it is expected that the activity of the Responder and/or one or more of the Distorters is affected. Depending on the type of alteration/abolishment of Responder/Distorter functions, these transgenic animals may be used in breeding schemes corresponding to the ones addressed above.
Finally, the present invention relates to a method for the distortion, to a non-Mendelian ratio, of the transmission of a genetic trait from male mammals to their offspring comprising expressing during spermatogenesis/spermiogenesis a gene involved in sperm motility and/or fertilization.
In a preferred embodiment of the invention said genetic trait determines the sex.
In another preferred embodiment of the method of the invention said gene is under the control of a promoter that allows expression during spermatogenesis/spermiogenesis.
The promoter may be the original promoter of said gene or may be derived from a different copy of said gene or from a different gene. Furthermore, the promoter may be derived from a copy of the homologous gene (in case more than one exists) from a different species or may be derived from a different gene from said different species. The promoters may also be modified in order to obtain optimum expression, which may be enhanced or reduced expression.
In a particularly preferred embodiment of the method of the invention said promoter allows the preferential or exclusive expression of said gene in sperm carrying said gene.
In a further preferred embodiment of the method of the invention said gene is engineered such as to interfere with the function of its wild type allele or with the function of other genes involved in sperm motility and/or fertilization, wherein said gene inhibits the function of one or more genes involved in sperm motility and/or fertilization, and/or wherein said gene causes cell death in spermatocytes/spermatids expressing it, and/or wherein said gene encodes a tag allowing the in vitro selection of sperm carrying said tag.
In a further preferred embodiment of the method of the invention said gene encodes an inhibitor of cAMP dependent protein kinase A.
In a particularly preferred embodiment said inhibitor is PKI or a functionally active derivative or fragment thereof.
As used in accordance with the present invention the term xe2x80x9cfunctionally active derivative or fragmentxe2x80x9d denotes molecules that deviate from PKI by one or more amino acid substitutions, deletions, and/or additions but essentially retain the biologically activity/activities of PKI, i.e. retain at least the inhibitory activity on cAMP dependent protein kinase A. Examples of functionally active derivatives or fragments of PKI are well known to the person skilled in the art and can be found, e.g., in catalogues of biotechnology companies (see, e.g., the Promega catalogue of 1998).
In another embodiment, the present invention relates to a transgenic animal comprising a gene as defined hereinabove.
Finally, the present invention relates to a sperm obtainable from the transgenic animal of the present invention.
The references cited in the present specification are herewith incorporated by reference.